Click-iT® Plus EdU for Flow Cytometry

The Click-iT® Plus EdU cell proliferation assay utilizes the power of click chemistry to provide a superior alternative to traditional BrdU methods for detecting and quantitating newly synthesized DNA. When the modified nucleoside, EdU (5-ethynyl-2′-deoxyuridine), is incorporated during DNA synthesis, it can be detected in a quick “click chemistry” reaction with minimal disruption to the cell.


Replace your cumbersome BrdU assays

Click-iT® Plus EdU—click chemistry simplicity makes it the faster, friendlier alternative to BrdU. Unlike assays using bromodeoxyuridine (BrdU), Click-iT® EdU assays are not antibody based and therefore do not require DNA denaturation for detection of the incorporated nucleoside. In addition, for increased utility, Click-iT® Plus can be multiplexed with fluorescent proteins like R-PE, R-PE tandems, and GFP.

The novel Click-iT® Plus EdU technology is:

  • Fast—detection in as little as 60 minutes
  • Accurate—no artifacts arising from the use of antibodies and denaturation steps
  • Multiplexable—compatible with R-PE (and tandems) and fluorescent proteins

When you compare the two methods side-by-side, the benefits of Click-iT® Plus EdU for cell proliferation are clear. (Table 1).

Table 1. Click-iT® Plus EdU vs. BrdU for cell proliferation.

  Click-iT® Plus EdU BrdU
Regents required for basic experiment
Number of steps
6
(No need for multiple permeabilization steps,
no need for DNase treatment)
~10
Time to results
(labeling+detection)
Typically <90 min
5 hours to overnight
DNA denaturation
None
Necessary
Multiplexable
Yes
Needs to be determined

The science behind Click-iT® Plus EdU Kits for flow cytometry

The Click-iT® Plus EdU advantage is in the chemistry—small, unique, and low-background labeling and detection moieties that react specifically and covalently with one another. 5-ethynyl-2´-deoxyuridine (EdU) is a nucleoside analog containing an alkyne. In a copper-catalyzed reaction, the alkyne reacts with a dye-labeled picolyl azide, forming a stable covalent bond. The small size of the azide reagent allows efficient access to the DNA without the need for harsh cell treatment. This simplifies the assay considerably, and the results you achieve are similar to (or better than) those typically achieved using BrdU (Figures 1 and 2).

Click-iT® Plus for Flow Cytometry
Alexa Fluor® 647 Click-iT® Plus EdU labeling with green fluorescent protein (GFP) and FxCycle™ Violet stain. GFP-expressing A375 (human malignant melanoma cells) were treated with 10 µM EdU for 2 hours and detected according to the recommended staining protocol. Panel A shows a histogram of cells labeled with Alexa Fluor® 647 picolyl azide, identifying cells in S-phase. Panel B shows an overlay histogram of GFP-expression Click-iT® Plus EdU Alexa Fluor® 647 picolyl azide (black line) and in the absence of copper (gray line), demonstrating GFP fluorescence is preserved in the click reaction. Panel C shows a dual parameter plot of Click-iT® Plus EdU Alexa Fluor® 647 and FxCycle™ Violet stain for DNA content; data and EdU data, cells that are co-positive are in S-phase.
Click-iT® Plus for Flow Cytometry
Figure 2. Alexa Fluor® 488 Click-iT® Plus EdU labeling with mCherry fluorescent protein and FxCycle™ Far Red stain. mCherry-expressing A549 cells were treated with 10 µM EdU for 2 hours and detected according to the recommended staining protocol. Panel A shows a histogram of cells labeled with Alexa Fluor® 488 picolyl azide, identifying cells in S-phase. Panels B and C show mCherry fluorescence from cells that were labeled with Click-iT® Plus EdU Alexa Fluor® 488 picolyl azide in the presence of copper (Panel B) and in the absence of copper (Panel C), demonstrating that the mCherry fluorescence is preserved in the click reaction. Panel D shows a dual parameter plot of Click-iT® Plus EdU Alexa Fluor® 488 and FxCycle™ Far Red stain for DNA content; cells that are co-positive are in S-phase.

Click-iT® Plus Assay Kits for flow cytometry

Sku Name Size Price Qty
C10632 Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit 50 assays USD 495.00
C10633 Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit 100 assays USD 850.00
C10634 Click-iT® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit 50 assays USD 495.00
C10635 Click-iT® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit 100 assays USD 850.00
C10636 Click-iT® Plus EdU Pacific Blue™ Flow Cytometry Assay Kit 50 assays USD 495.00